Drug Screening And The Mechanism Study For Promoting Oligodendroglial Differentiation And Remyelination

Oligodendrocytes(OLs)are the myelin forming cells in the central nervous system(CNS),differentiated from the oligodendrocyte precursor cells(OPCs).Myelin is indispensable for fast and effective electrical transduction;besides,it provides metabolic supports for axons.But oligodendroglial cells are relatively sensitive to numerous intrinsic and extrinsic damaging factors,which may ultimately cause cell death and demyelination.Damage or malfunction of oligodendrocytes can lead to serious diseases,like multiple sclerosis(MS),or Schizophrenia.While OPCs in adults maintained their abilities to differentiate and myelinate axons to restore myelin sheaths,namely remyelination when necessary.But under pathological conditions,the remyelination process is often incomplete and insufficient.Therefore,it is essential to find an approach to identify compounds that can efficiently promote OPC differentiation and remyelination for the treatment of demyelination diseases.An efficient platform would be critical for screening potent remyelination drugs and it would also facilitate us to identify the targets of individual drugs.The traditional method to verify drugs or compounds in promoting the differentiation of OPCs in cultures is time-consuming and inconsistent.Besides,it's difficult to compare the effects between the tested drugs or compounds and pick out the best one.Therefore,it is necessary to establish a new screening method that can fulfill the need to screen the drugs efficiently and rapidly.A series of studies have revealed that high-throughput techniques can provide an efficient approach to identify effective drugs/compounds and the underlying mechanisms.The Binary indicant for myelination using Micropillar Arrays(BIMA),is capable of screening myelination-promoting drugs in a high-throughput manner.This technique allows us to obtain much more informative details of the biological process and more available and completed information on the gene expression in these biological processes and help us to better understand the mechanisms.Cooporating with labs abroad,we have successful established the BIMA screening system.In our study,we applied the high throughput screening BIMA system to screen hundreds of compounds.Among the myelination-promoting drugs or compounds selected by BIMA screeing,we found that,quetiapine(QUE),an atypical antipsychotic drug may be a competent candidate to promote OPC differentiation and remyelination.Furthermore,we studied the underlying mechanism of QUE with microarray by examining significant genome changes and featured gene ontological changes after QUE treatment.The result suggested that chromatin remodeling related genes may have played essential roles in OPC differentiation.Recent research results suggested that epigenetics,especially featuring chromatin remodeling complex core subunit Brg1,has been actively involved in OPC differentiation.Thus,we designed and set up an oligodendroglial specific transgenetic mice line,and made preliminary studies on the function of BAF155,which is a molecule that may have essential regulatory roles in OPC differentiation.On the other hand,we also examined the role of aspirin(ASA),one of the candidates for promoting OPC differentiation and myelination screened by BIMA.We demonstrated that ASA can promote OPC differentiation,likely by inhibition of Wnt signaling.Main results are listed as below:(1)Screening myelination-enhancing drugs by using BIMA.We applied most recent high-throughput screen system for myelination,BIMA.By filtering from 200 compounds or drugs in our storage bank,we found several drugs that can promote OPC differentiation.Using immunofluorescence and Western Blot,we testified basic cellular effect of the drugs we are interested in,including clemastine,U50488 and quetiapine(QUE).Among these,the most attractive is the untypical antipsychotic drug QUE,which our lab has been long been focusing in preliminary studies.(2)Verification of Que in promoting OPC differentiation and remyelinationBy immunofluorescence and Western Blot,we found that QUE treatment not only can promote the differentiation and maturation of OPCs,but also significantly increased the expression of MBP in the corpus callosum region of the newborn rats.In demyelination model set up with Cuprizone,QUE treatment strongly promoted remyelinatio,indicating that QUE is a ponent candidate drug,and it can be used as a tool to study the underlying mechanisms of promoting the differentiation of OPCs and remyelination and searching for new targets for the drugs.(3)Identification of BAF155 as a regulatory factor for OPC differentiation.Using QUE as a tool for microarray screening,we made a survey of the underlying mechanism.First,we figured out significantly altered genes in OPC normal differentiation process with microarray,thus we confirmed that the technique can correctly and more thoroughly reflect gene changes and signaling pathway patterns in normal differentiation process.Successively,by comparing with the normal differentiation process,we clustered significantly up/down-regulated genes after QUE treatment,and the result indicated that chromatin remodeling related genes were significantly down-regulated,suggesting their important roles in OPC differentiation.By analyzing specific genes belonging to the chromatin remodeling cluster,we found that the most significant altered gene is BAF155,therefore,it may be an essential regulatory molecule in OPC differentiation.(4)BAF155 functions in regulating oligodendroglial differentiationWe designed and set up a BAF155-flox mice line.By hybridizing with mice characterized of Cre in oligodendroglial lineage specific marker PDGFaR,we successfully acquired a conditional knockout mouse(BAF155 fl/fl,PDGFaRCreERT)by inducing knockout with Tamoxifen.Successively,we applied immunofluorescence and in-situ hybridization to study the effect of BAF155 on OPC development.The result revealed that knocking out BAF155 in OPC will result in a down-regulation of mature OL marker MBP and MAG,indicating that BAF155 played essential roles in OPC differentiation,and its effect is not simply inhibitive.Further study needs to be done to reveal the detailed mechanism.(5)Identification of Asprin as a novel drug for promoting OPC differentiationIn OPC culture,we found that,cell number of double labeling of Ki67~+/PDGFaR~+OPCs(proliferating cells)showed no significant difference between the vehicle and ASA treatment group(p>0.05),while the cell number of MBP~+OLs was significantly higher in ASA treatment group than the vehicle group.These cells were also morphologically more mature,judging from an increased number of longer processes.In white matter development model of newborn rats,after ASA treatment,MBP expression was up-regulated.Further study has revealed that after ASA treatment,important signal molecule in Wnt/?-catenin signaling pathway,GSK-3?,was up-regulated.Moreover,the phosphorylation level of its downstream target?-catenin up-regulated while the phosphorylation level of GSK-3?was down-regulated.A selective Wnt/?-catenin signaling pathway synergist,QS11 treatment resulted in OPC differentiation inhibition,while ASA can neutralize the effect.These results indicated that,ASA may promote the differentiation of OPCs by inhibiting Wnt/?-catenin signaling pathway while it has no effect on proliferation.In all,in order to find an effective target to promote myelination,we applied high-throughput techniques like BIMA and microarray,applying these techniques,we figured out that QUE is an effective candidate.Using QUE as a tool drug,we showed that chromatin remodeling related genes played essential roles in OPC differentiation.We also demonstrated that ASA can promote OPC differentiation by inhibition of Wnt/?-catenin signaling.Our study results may provide novel perspectives for remyelination.